Individual plasma proteins detected on rough biomaterials by phase imaging AFM

In the past several years, atomic force microscopy (AFM) has provided topographic images of adsorbed plasma proteins in situ at unprecedented resolution. Imaging has been limited to adsorbed protein on relatively smooth model substrates such as mica, graphite, or self-assembled monolayers on which the small height of the protein can be observed from the background. The inherent roughness of biomaterial surfaces has prevented observation of adsorbed proteins in topographic images. We report imaging isolated fibrinogen molecules adsorbed on National Heart Lung and Blood Institute (NHLBI) reference materials polydimethylsiloxane and low-density polyethylene in situ using phase imaging AFM. Fibrinogen, a plasma protein important for blood coagulation and platelet aggregation, was adsorbed from dilute solution onto reference biomaterial surfaces at sub-monolayer coverage. Tapping mode AFM was used to image the samples. For polydimethylsiloxane, the lateral size of the surface features is much greater than the dimensions of proteins. This allowed adsorbed proteins to be observed in topographic images. The phase imaging signal of tapping mode AFM provides information on differences in material properties of the surface, and was used to distinguish individual protein molecules from the underlying polymer surface. On the low-density polyethylene surface, characteristic topographical features are of the same magnitude as the protein molecules, so that protein cannot be distinguished from the surface using topographic images. However, phase images were used to successfully locate and characterize the distribution of the protein. Phase imaging was not able to distinguish fibrinogen adsorbed onto expanded polytetrafluoroethylene. The utility and limitations of the phase imaging technique for characterizing protein adsorption to rough surfaces is discussed.